Efforts are focused in two distinct, yet interrelated areas. One goal is to define how the generation of thrombin at the human platelet surface is effected and regulated. Our second goal is to begin to define how thrombin, once formed, interacts with platelet proteins to modulate its activity and the fibrinolytic response. Since thrombin is generated through the proper assembly and function of Prothrombinase at the platelet surface we will test several hypotheses relevant to how functional complex assembly occurs and how the platelet actively regulates these processes. Quantitation of the coordinate binding of factors Va and Xa to platelets followed by assessment of their functional activity under a variety of conditions will allow us to the test the following hypotheses: 1) that Effector Protease Receptor 1 (EPR-1) functions as part of the Prothrombinase receptor(s) at the activated platelet surface; 2) that receptor expression and functional Prothrombinase assembly can be modulated by platelet adherence to extracellular matrix proteins and 3) agonist-induced release and binding of platelet factor V9a) plays a preeminent role in Prothrombinase regulation since platelet factor Va is functionally (and perhaps structurally) different than plasma factor Va with respect to inactivation by proteases such as APC, plasmin, elastase and cathepsin G. Finally, we hypothesize that the activated platelet continues to promote a procoagulant response by inhibiting fibrinolysis. To test this hypothesis we will determine if the fibrinolytic response is prolonged through the release of factor Va from the activated platelet as well as the expression of thrombomodulin since both molecules will enhance, albeit through different mechanisms, the thrombin-catalyzed activation of the thrombin activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase B-like molecule. Our accomplishments included demonstrating that: 1) the defect in the bleeding disorder termed Factor V Quebec is due to proteolysis of the cofactor pool stored within the platelet a-granule; 2) activated protein C (APC) promotes fibrinolysis by regulating the activation of TAFI; 3) the prothrombotic effect associated with procoagulation factor mutant, Factor V Leiden, which is resistant to inactivation by APC, is due in part to its sustained generation of thrombin and activation of TAFI leading to sustained and prolonged thrombus formation. Our current plans are: 1) to identify and characterize the thrombin high affinity binding site on the platelet membrane surface; 2) to continue to define the functional significance of the platelet membrane protein glycoprotein Ib as it relates to the platelet high affinity binding site; 3) to define the mechanisms by which activated protein C, plasmin and elastase catalyze the activation and/or inactivation of platelet factors V and Va; 4) to identify and characterize the thrombomodulin-like molecule on the platelet membrane surface, and 5) to develop protocols for the isolation of homogeneous platelet factor V/Va for biochemical characterization and comparison to the plasma- derived protein.